siRNA / miRNA gene silencing Human - Primary Human Hepatocytes CYP3A4

miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time consuming, but provide a more permanent expression of RNAi in the cells, and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines. When using oligos, the ideal concentration lies between 10-50nM for effective transfection.

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ON-TARGETplus Human CYP3A4 (1576) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	Using siRNA to suppress CYP proteins was conducted by modification of the method published by Vozza-Brown et al. [37]. After 5 h incubation with hepatocyte plating medium, cells were washed and treated with Dharmacon ON-TARGETplus human CYP3A4 or CYP2B6 SMARTpool siRNA. Positive ON-TARGETplus GAPD human control pool and negative control ON-TARGETplus non-targeting pool were also included (Table 2). A non-siRNA treated control was also included to differentiate between the loss of gene expression due to endogenous degradation and loss due to siRNA treatment. 1.25 μL of transfection reagent Lipofectamine RNAiMAX™ was complexed with 50 or 100 nM ON-TARGETplus CYP3A4 or CYP2B6 siRNA, respectively, in reduced serum opti-MEM® I medium for 30 min prior to addition to cell culture. Hepatocytes were exposed to siRNA overnight for 15 h before the cells were washed and replaced with standard hepatocyte maintenance medium. Cells were subsequently incubated at 37 °C with 5% (v/v) CO2 for 48, 72 and 96 h post CYP3A4 siRNA treatment, and for 12, 24, 36, 48 and 60 h after CYP2B6 siRNA treatment. CYP3A4 and CYP2B6 metabolic activity, mRNA and protein expression were assessed at these time points. Time 0 h was taken after 5 h incubation with plating media and prior to siRNA transfection and maintenance media replacement. Following siRNA transfection, maintenance media was replaced every 24 h.

Protocol tips
Using siRNA to suppress CYP proteins was conducted by modification of the method published by Vozza-Brown et al. [37]. After 5 h incubation with hepatocyte plating medium, cells were washed and treated with Dharmacon ON-TARGETplus human CYP3A4 or CYP2B6 SMARTpool siRNA. Positive ON-TARGETplus GAPD human control pool and negative control ON-TARGETplus non-targeting pool were also included (Table 2). A non-siRNA treated control was also included to differentiate between the loss of gene expression due to endogenous degradation and loss due to siRNA treatment. 1.25 μL of transfection reagent Lipofectamine RNAiMAX™ was complexed with 50 or 100 nM ON-TARGETplus CYP3A4 or CYP2B6 siRNA, respectively, in reduced serum opti-MEM® I medium for 30 min prior to addition to cell culture. Hepatocytes were exposed to siRNA overnight for 15 h before the cells were washed and replaced with standard hepatocyte maintenance medium. Cells were subsequently incubated at 37 °C with 5% (v/v) CO2 for 48, 72 and 96 h post CYP3A4 siRNA treatment, and for 12, 24, 36, 48 and 60 h after CYP2B6 siRNA treatment. CYP3A4 and CYP2B6 metabolic activity, mRNA and protein expression were assessed at these time points. Time 0 h was taken after 5 h incubation with plating media and prior to siRNA transfection and maintenance media replacement. Following siRNA transfection, maintenance media was replaced every 24 h.

Protocol tips

Using siRNA to suppress CYP proteins was conducted by modification of the method published by Vozza-Brown et al. [37]. After 5 h incubation with hepatocyte plating medium, cells were washed and treated with Dharmacon ON-TARGETplus human CYP3A4 or CYP2B6 SMARTpool siRNA. Positive ON-TARGETplus GAPD human control pool and negative control ON-TARGETplus non-targeting pool were also included (Table 2). A non-siRNA treated control was also included to differentiate between the loss of gene expression due to endogenous degradation and loss due to siRNA treatment. 1.25 μL of transfection reagent Lipofectamine RNAiMAX™ was complexed with 50 or 100 nM ON-TARGETplus CYP3A4 or CYP2B6 siRNA, respectively, in reduced serum opti-MEM® I medium for 30 min prior to addition to cell culture. Hepatocytes were exposed to siRNA overnight for 15 h before the cells were washed and replaced with standard hepatocyte maintenance medium. Cells were subsequently incubated at 37 °C with 5% (v/v) CO2 for 48, 72 and 96 h post CYP3A4 siRNA treatment, and for 12, 24, 36, 48 and 60 h after CYP2B6 siRNA treatment. CYP3A4 and CYP2B6 metabolic activity, mRNA and protein expression were assessed at these time points. Time 0 h was taken after 5 h incubation with plating media and prior to siRNA transfection and maintenance media replacement. Following siRNA transfection, maintenance media was replaced every 24 h.

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